Targeting the Mitotic Checkpoint to Kill Tumor Cells

One of the most common hallmarks of cancer cells is aneuploidy or an abnormal number of chromosomes. This abnormal chromosome content is a consequence of chromosome missegregation during mitosis, a defect that is seen more frequently in tumor cell divisions as in normal cell divisions. In fact, a large fraction of human tumors display a chromosome instable phenotype, meaning that they very frequently missegregate chromosomes. This can cause variegated aneuploidy within the tumor tissue. It has been argued that this hallmark of cancer could be exploited in anti-cancer therapies. Here we test this hypothesis by inactivation of the mitotic checkpoint through RNAi-mediated depletion of an essential checkpoint component, Mps1. The mitotic checkpoint delays segregation of chromosomes during mitosis until all chromosomes are properly attached to the mitotic spindle. Its inactivation will therefore lead to increased segregation errors. Indeed, we show that this can lead to increased cell death in tumor cells. We demonstrate that increased cell death is associated with a dramatic increase in segregation errors. This suggests that inhibition of the mitotic checkpoint might represent a useful anti-cancer strategy

Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs

The DNA-dependent adenosine triphosphatase (ATPase) Plk1-interacting checkpoint helicase (PICH) has recently been implicated in spindle checkpoint (SAC) signaling (Baumann et al., Cell 128(1):101–114, 2007). Depletion of PICH by siRNA abolished the SAC and resulted in an apparently selective loss of Mad2 from kinetochores, suggesting a role for PICH in the regulation of the Mad1–Mad2 interaction. An apparent rescue of SAC functionality by overexpression of PICH in PICH-depleted cells initially seemed to confirm a role for PICH in the SAC. However, we have subsequently discovered that all PICH-directed siRNA oligonucleotides that abolish the SAC also reduce Mad2 mRNA and protein expression. This reduction is functionally significant, as PICH siRNA does not abolish SAC activity in a cell line that harbors a bacterial artificial chromosome driving the expression of murine Mad2. Moreover, we identified several siRNA duplexes that effectively deplete PICH but do not significantly affect SAC functionality or Mad2 abundance or localization. Finally, we discovered that the ability of overexpressed PICH to restore SAC activity in PICH-depleted cells depends on sequestration of the mitotic kinase Plk1 rather than ATPase activity of PICH, pointing to an underlying mechanism of “bypass suppression.” In support of this view, depletion or inhibition of Plk1 also rescued SAC activity in cells harboring low levels of Mad2. This observation suggests that a reduction of Plk1 activity partially compensates for reduced Mad2 levels and argues that Plk1 normally reduces the strength of SAC signaling. Collectively, our results question the role of PICH in the SAC and instead identify Mad2 as a sensitive off target for small RNA duplexes. In support of the latter conclusion, our evidence suggests that an off-target effect on Mad2 may also contribute to explain the apparent role of the Tao1 kinase in SAC signaling (Draviam et al., Nat Cell Biol 9(5):556–564, 2007)

A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease

Chromosomal Instability by Inefficient Mps1 Auto-Activation Due to a Weakened Mitotic Checkpoint and Lagging Chromosomes

BACKGROUND: Chromosomal instability (CIN), a feature widely shared by cells from solid tumors, is caused by occasional chromosome missegregations during cell division. Two of the causes of CIN are weakened mitotic checkpoint signaling and persistent merotelic attachments that result in lagging chromosomes during anaphase. PRINCIPAL FINDINGS: Here we identify an autophosphorylation event on Mps1 that is required to prevent these two causes of CIN. Mps1 is phosphorylated in mitotic cells on at least 7 residues, 4 of which by autophosphorylation. One of these, T676, resides in the activation loop of the kinase domain and a mutant that cannot be phosphorylated on T676 is less active than wild-type Mps1 but is not kinase-dead. Strikingly, cells in which endogenous Mps1 was replaced with this mutant are viable but missegregate chromosomes frequently. Anaphase is initiated in the presence of misaligned and lagging chromosomes, indicative of a weakened checkpoint and persistent merotelic attachments, respectively. CONCLUSIONS/SIGNIFICANCE: We propose that full activity of Mps1 is essential for maintaining chromosomal stability by allowing resolution of merotelic attachments and to ensure that single kinetochores achieve the strength of checkpoint signaling sufficient to prevent premature anaphase onset and chromosomal instability. To our knowledge, phosphorylation of T676 on Mps1 is the first post-translational modification in human cells of which the absence causes checkpoint weakening and CIN without affecting cell viability

p68/DdX5 supports β-Catenin & RNAP II during androgen receptor mediated transcription in prostate cancer

The DEAD box RNA helicase p68 (Ddx5) is an important androgen receptor (AR) transcriptional co-activator in prostate cancer (PCa) and is over-expressed in late stage disease. β-Catenin is a multifunctional protein with important structural and signalling functions which is up-regulated in PCa and similar to p68, interacts with the AR to co-activate expression of AR target genes. Importantly, p68 forms complexes with nuclear β-Catenin and promotes gene transcription in colon cancer indicating a functional interplay between these two proteins in cancer progression. In this study, we explore the relationship of p68 and β-Catenin in PCa to assess their potential co-operation in AR-dependent gene expression, which may be of importance in the development of castrate resistant prostate cancer (CRPCa). We use immunoprecipitation to demonstrate a novel interaction between p68 and β-Catenin in the nucleus of PCa cells, which is androgen dependent in LNCaP cells but androgen independent in a hormone refractory derivative of the same cell line (representative of the CRPCa disease type). Enhanced AR activity is seen in androgen-dependent luciferase reporter assays upon transient co-transfection of p68 and β-Catenin as an additive effect, and p68-depleted Chromatin-Immunoprecipitation (ChIP) showed a decrease in the recruitment of the AR and β-Catenin to androgen responsive promoter regions. In addition, we found p68 immunoprecipitated with the processive and non-processive form of RNA polymerase II (RNAP II) and show p68 recruited to elongating regions of the AR mediated PSA gene, suggesting a role for p68 in facilitating RNAP II transcription of AR mediated genes. These results suggest p68 is important in facilitating β-Catenin and AR transcriptional activity in PCa cells

Quantitative Mass Spectrometry Analysis Reveals Similar Substrate Consensus Motif for Human Mps1 Kinase and Plk1

Background Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known. Methodology/Principal Findings Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus. Conclusions/Significance hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase

Organization of multiprotein complexes at cell–cell junctions

The formation of stable cell–cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell–cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell–cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell–cell contact formation and maintenance

Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability